3. 3 cc, 2.88
00 , clear Nordson
® EFD
® 7012072 syringe barrel with
piston and gas adapter assembly.
4. PURAC
® medical grade poly(ε-caprolactone).
2.2 Cell Culture
Here, we have used primary human-derived dermal fibroblasts.
Similar work has been performed with 3T3 fibroblasts, mesothelial
and mesenchymal stromal cells, keratinocytes, and osteoblasts.
1. Tissue culture flasks.
2. Twelve-well plates.
3. Falcon tubes.
4. Hemocytometer.
5. Fibroblasts growth medium: Complete Dulbecco’s Modified
Eagle Medium (DMEM). To 500 mL of DMEM, add 5 mL of
L-glutamine, 5 mL of penicillin-streptomycin, and 50 mL of
fetal calf serum. Store at 4
C.
6. Sterile phosphate-buffered saline (PBS): without magnesium
and calcium.
7. Trypsin-EDTA: 0.05% (Gibco, Thermo Fisher Scientific).
8. Trypan blue.
2.3 Visualization
1. Glutaraldehyde: 3% in cacodylate buffer.
2. 1 M cacodylate buffer.
3. Osmium tetroxide solution: 1% osmium tetroxide in cacodylate
buffer.
4. Ethanol/water solutions: 40%, 50%, 70%, 90%, and 100%.
5. Hexamethyldisilazane (HMDS).
6. Paraformaldehyde (PFA): 4% in PBS, supplemented with magnesium and calcium.
7. PBS supplemented with magnesium and calcium: 100 mg of
calcium chloride (CaCl 2 ) and 100 mg of magnesium chloride
(MgCl 2 ·6H 2 O) in 1 L of PBS.
8. Permeabilizing buffer: 0.2% Triton X-100 in PBS.
9. Blocking solution: 1% bovine serum albumin (BSA) in PBS.
10. Staining solution: Add 0.8 U/mL FITC-conjugated phalloidin
and 5 μg/ML DAPI to blocking solution.
3 Methods
3.1 Fabricating the
Scaffolds
1. Tightly pack between 1 and 2 g of the PCL polymer pellets into
a 3 cc, 2.88
00 syringe barrel, and attach the 23GA, 1
00 needle.
Place the plastic gas piston over the end of the syringe, and
Melt Electrospinning Writing
115
00 , clear Nordson
® EFD
® 7012072 syringe barrel with
piston and gas adapter assembly.
4. PURAC
® medical grade poly(ε-caprolactone).
2.2 Cell Culture
Here, we have used primary human-derived dermal fibroblasts.
Similar work has been performed with 3T3 fibroblasts, mesothelial
and mesenchymal stromal cells, keratinocytes, and osteoblasts.
1. Tissue culture flasks.
2. Twelve-well plates.
3. Falcon tubes.
4. Hemocytometer.
5. Fibroblasts growth medium: Complete Dulbecco’s Modified
Eagle Medium (DMEM). To 500 mL of DMEM, add 5 mL of
L-glutamine, 5 mL of penicillin-streptomycin, and 50 mL of
fetal calf serum. Store at 4
C.
6. Sterile phosphate-buffered saline (PBS): without magnesium
and calcium.
7. Trypsin-EDTA: 0.05% (Gibco, Thermo Fisher Scientific).
8. Trypan blue.
2.3 Visualization
1. Glutaraldehyde: 3% in cacodylate buffer.
2. 1 M cacodylate buffer.
3. Osmium tetroxide solution: 1% osmium tetroxide in cacodylate
buffer.
4. Ethanol/water solutions: 40%, 50%, 70%, 90%, and 100%.
5. Hexamethyldisilazane (HMDS).
6. Paraformaldehyde (PFA): 4% in PBS, supplemented with magnesium and calcium.
7. PBS supplemented with magnesium and calcium: 100 mg of
calcium chloride (CaCl 2 ) and 100 mg of magnesium chloride
(MgCl 2 ·6H 2 O) in 1 L of PBS.
8. Permeabilizing buffer: 0.2% Triton X-100 in PBS.
9. Blocking solution: 1% bovine serum albumin (BSA) in PBS.
10. Staining solution: Add 0.8 U/mL FITC-conjugated phalloidin
and 5 μg/ML DAPI to blocking solution.
3 Methods
3.1 Fabricating the
Scaffolds
1. Tightly pack between 1 and 2 g of the PCL polymer pellets into
a 3 cc, 2.88
00 syringe barrel, and attach the 23GA, 1
00 needle.
Place the plastic gas piston over the end of the syringe, and
Melt Electrospinning Writing
115